Use of prostate tumor inducing gene for detection of cancer cells

ABSTRACT

This invention provides a method for detecting cancer cells in a sample comprising detection of the expression of a Prostate Tumor Inducing Gene in the sample, wherein a positive detection of the expression indicates the presence of cancer cells in the sample. In an embodiment, the expression is performed by measuring the level of PTI mRNA. The mRNA is measured by reverse transcription-polymerase chain reaction using at least one pair of approporiate primers. It is well known in the art that once the sequence of the Prostate Tumor Inducing Gene is determined, an appropriate pair of primers may be selected. This invention also provides the above method, wherein the expression is performed by measuring the level of PTI protein. The level of PTI protein is measured by steps of: a) contacting the sample with an antibody capable of specifically recognizing PTI-1 protein under conditions permitting formation of complexes between the PTI-1 protein and the antibody; and b) measuring the complex formed, thereby measuring the level of PTI-1 protein expressed in the cancer cells.

[0001] This application is a continuation-in-part application of U.S.Ser. No. 08/708,208, filed Sep. 6, 1997, the content of which isincorporated into this application by reference.

BACKGROUND OF THE INVENTION

[0002] Rapid expression cloning and differential RNA display identifiesa gene, prostate tumor inducing gene-1 (PTI-1), that is differentiallyexpressed in prostate cancer versus normal prostate and benign prostatichypertrophy. PTI-1 encodes a truncated and mutated human elongationfactor 1α (EF-1α), and its 5′ untranslated (UTR) region sharessignificant homology with the 23S ribosomal RNA gene of Mycoplasmahyopneumoniae. PCR with human genomic DNAs, using PTI-1 5′ UTR specificprimers, suggests that this sequence is part of the human genome.Furthermore, RT-PCR, with one primer specific to the 5′ UTR region andthe other to the EF-1α coding region, amplifies PTI-1 transcripts fromtotal RNA of various human tumor cell lines and blood samples fromprostate carcinoma patients. RT-PCR products with the predicted size andsequence of PTI-1 are detected in RNAs from cell lines of humanprostate, breast and colon carcinomas. This RT-PCR product is shown bySouthern blotting and sequence analyses to contain the junction sequencebetween the 5′ UTR and the coding region of the PTI-1 gene. Furthermore,RT-PCR analysis indicates that the PTI-1 gene is also expressed inprostate carcinoma patient derived blood samples. On the basis of serialdilution experiments, PTI-1 can detect 1 prostate carcinoma cell in 10⁸cells not expressing PTI-1. In this context, PTI-1 represents the mostsensitive marker currently available for detecting human prostatecancer. This study confirms the authenticity of the PTI-1 gene anddocuments its potential clinical utility as a sensitive and specificindicator of prostate cancer progression.

[0003] Adenocarcinoma of the prostate is presently the most prevalentinternal cancer of men in the United States and the second most frequentcause of cancer-related deaths. Current methodologies for the earlydetection of prostate cancer, including physical examination, monitoringPSA³ levels, tissue biopsy, ultrasound and bone scans, are restricted inboth sensitivity and specificity (1-3). In addition, present testingmodalities do not permit a distinction between cancers that will remainindolent and those which will prove aggressive and life threatening(1-4). Using DNA transfection approaches with a novel acceptor cellline, CREF-Trans 6 (5), and the molecular approach of differential RNAdisplay (6), a novel putative prostatic carcinoma tumor inducingoncogene, PTI-1, has been identified and cloned from a human prostatecarcinoma, LNCaP, cDNA library (7). Using RT-PCR approaches with primerscorresponding to the 5′ UTR region of PTI-1, expression is detected in15 of 16 carcinomas of the prostate, but not in normal prostate or BPHtissue (7). Although further testing with a larger number of patientsamples is clearly needed, these provocative results suggest that PTI-1monitoring might prove beneficial in prostate cancer diagnostics.

[0004] The full-length PTI-1 cDNA is 2,123 bp and it encodes a truncatedand mutated human EF-1α (7) (FIG. 1). The structure of the PTI-1 cDNA isunique in that its 5′ UTR shares significant homology (approximately85%) with the prokaryotic 23S ribosomal RNA gene from Mycoplasmahyopneumoniae. This high degree of sequence homology between the 5′ UTRof the PTI-1 gene and prokaryotic 23S ribosomal RNA gene raises concernsthat contamination by bacteria in the LNCaP cell culture used to preparethe cDNA library and subsequent cloning artifacts may be responsible forthe identification of the PTI-1 gene. Confirming the authenticity of thePTI-1 gene is mandatory before further studies can be conducted toelucidate any potential role of PTI-1 in human prostate cancerdevelopment and evolution.

[0005] In the present study, the question of the validity of the PTI-1gene was addressed by analyzing its presence in the human genome,transcripts in tumor cell lines and presence in blood samples frompatients with prostate cancer. The results of these investigationsdemonstrate definitively that the identification of the PTI-1 gene isunlikely due to bacterial contamination and/or technical artifacts.Moreover, PTI-1 gene expression may provide an extremely sensitivemarker for prostate carcinoma progression as reflected by the presenceof prostate carcinoma cells in a patients' bloodstream.

SUMMARY OF THE INVENTION

[0006] This invention provides a method for detecting cancer cells in asample comprising detection of the expression of a Prostate TumorInducing Gene in the sample, wherein a positive detection of theexpression indicates the presence of cancer cells in the sample.

[0007] In an embodiment of this invention, the Prostate Tumor InducingGene is PTI-1. In a separate embodiment, the Prostate Tumor InducingGene is PTI-2. In another embodiment, the Prostate Tumor Inducing Geneis PTI-3.

[0008] In an embodiment, the expression is performed by measuring thelevel of PTI mRNA. The mRNA is measured by reversetranscription-polymerase chain reaction using at least a pair ofappropriate primers. It is well known in the art that once the sequenceof the Prostate Tumor Inducing Gene is determined, appropriate pair ofprimers may be selected.

[0009] In an embodiment, at least one of the primers is complementary toeither the 5 prime or 3 prime untranslated region. In a furtherembodiment, the primers are complementary to the 5 prime untranslatedregion. In a still further embodiment, one of the primers iscomplementary to the 5 prime untranslated region and the other primer iscomplementary to the coding region. In another embodiment, the primersare complementary to the 3 prime untranslated region. In separateembodiment, one of the primers is complementary to the 3 primeuntranslated region and the other primer is complementary to the codingregion.

[0010] This invention also provides the above method, wherein theexpression is performed by measuring the level of PTI protein. The levelof PTI protein is measured by steps of: a) contacting the sample withantibody capable of specifically recognizing PTI-1 protein underconditions permitting formation of complexes between the PTI-1 proteinand the antibody; and b) measuring the complex formed, thereby measuringthe level of PTI-1 protein expressed in the cancer cells.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1: Structure of the PTI-1 gene and primers and probesequences used for analysis. (A) Schematic diagram of the PTI-1 cDNAstructure. The 5′ and 3′ UTRs are represented by thin lines, and thecoding region is represented by a hatched box. The thick line representsthe bridge specific probe (BSP) used to analyze the junction regionbetween the 5′ UTR and the coding region. Arrow heads indicate theposition of PCR and RT-PCR primers. (B) Sequences of PCR and RT-PCRprimers and probe used in Southern blotting analysis.

[0012]FIG. 2: The 5′ UTR sequence of the PTI-1 gene is present in thehuman genome. One μg of human cerebellum genomic DNA is amplified withprimer pairs UU and UL (see FIG. 1). A specific PCR product with theanticipated size of 424 bp is generated, which is not affected by RNaseAtreatment. This product is not generated following removal of either oneof the primers, DNA template, or Taq polymerase. MW, molecular weightmarker, is 100 bp DNA ladder (GibcoBRL).

[0013]FIG. 3: PTI-1 gene expression in human tumor cell lines. (A) Aspecific RT-PCR product with the expected size is generated by primerpairs BU and BL (see FIG. 1) in human prostate, breast and coloncarcinoma cell line total RNAs. This product is not present inCREF-Trans 6 cells, but it is present in CREF-Trans 6:4 NMT cells (nudemouse tumor-derived CREF-Trans 6 cells transfected with LNCaP DNA). (B)This RT-PCR product hybridizes with an oligonucleotide probe (BSP) usingstringent hybridization conditions. The BSP consists of 10 nt on eitherside of the junction point between the 5′ UTR and the coding region ofthe PTI-1 gene (see FIG. 1). (C) The same pattern of expression isdetected when the 5′ UTR specific primer pair UU and UL are used (seeFIG. 1).

[0014]FIG. 4: Sensitivity of PTI-1 in detecting prostate carcinoma cellsin diluted cell culture samples and in patient blood samples. (A)Ethidium bromide-stained gel of PCR products generated using PSA (14)and PTI-1 5′ UTR (7) specific primers in LNCaP cells diluted withCREF-Trans 6 cells. (B) RT-PCR analysis of PTI-1 expression in bloodsamples from normal males, normal females and prostate cancer patientswith stage D disease. The PCR amplified products generated using a PTI-15′ UTR primer pair (7) were blotted on nylon membranes and probed with a³²P-labeled DNA fragment of PTI-1. Specific samples were also analyzedby RT-PCR for expression of either PSA or PSM. N=normal; M=male;F=female; DU-145, human prostate carcinoma cell line; N.T.=not tested;+=expression; −=no expression; top number=patient code; D2 andD3=patients with stage D disease.

[0015]FIG. 5: RT-PCR of GAPDH, samples are treated according to thedescription in the Second Series of Experiment. Both GAPDH and theBridge region RT-PCT involved one round of amplification. Two round ofamplification (30 cycles/round) was required to achieve a signal withthe 5′ UTR primers.

[0016]FIG. 6 RT-PCR of Bridge region (BU and BL)

[0017]FIG. 7 RT-PCR of 5′UTR

DETAILED DESCRIPTION OF THE INVENTION

[0018] This invention provides a method for detecting cancer cells in asample comprising detection of the expression of a Prostate TumorInducing Gene in the sample, wherein a positive detection of theexpression indicates the presence of cancer cells in the sample. In anembodiment, the cancer cells are carcinoma cells. The cancer cellsinclude but are not limited to prostrate cancer cells, breast cancercells, colon cancer cells and lung cancer cells.

[0019] In an embodiment, the sample is a blood sample. In a separateembodiment, the sample is a urine sample. In a further embodiment, thesample is a semen sample.

[0020] In an embodiment of this invention, the Prostate Tumor InducingGene is PTI-1. In a separate embodiment, the Prostrate Tumor InducingGene is PTI-2. In another embodiment, the Prostrate Tumor Inducing Geneis PTI-3. PTI-1, -2 (SEQ ID NO: 10) and -3 (SEQ ID NO: 11), filed Jan.11, 1995 and Patent Cooperation Treaty Application No. PCT/US96/00307,filed January 1996.

[0021] In an embodiment, the expression is performed by measuring thelevel of PTI mRNA. The mRNA is measured by reversetranscription-plymerase chain reaction using at least a pair ofappropriate primers. It is well known in the art that once the sequenceof the Prostrate Tumor Inducing Gene is determined, appropriate pair ofprimers may be selected.

[0022] In an embodiment, at least one of the primers is complementary toeither the 5 prime or 3 prime untranslated region. In a furtherembodiment, the primers are complementary to the 5 prime untranslatedregion. In a still further embodiment, one of the primers iscomplementary to the 5 prime untranslated region and the other primer iscomplementary to the coding region. In another embodiment, the primersare complementary to the 3 prime untranslated region. In separateembodiment, one of the primers is complementary to the 3 primeuntranslated region and the other primer is complementary to the codingregion.

[0023] This invention further provides the above method wherein at leastone of the primers is selected from a group consisting of5′GAGTCTGAATAGGGCGACTT3′, 5′AGTCAGTACAGCTAGATGCC3′,5′ACCCGAGAGGGGAGTGAAATA 3′, 5′TGCCGCCATTCCACATTCAGT3′,5′ATGGGGGTAGAGCACTGAATG3′, 5′AACACCAGCAGCAACAATCAG3′ and5′AAATTAAGCTATGCAGTCGG3′. Since the complete sequence of some ProstateTumor Inducing gene has been known, other appropriate primer may easilybe selected.

[0024] This invention also provides the above method, wherein theexpression is performed by measuring the level of PTI protein. The levelof PTI protein is measured by steps of: a) contacting the sample withantibody capable of specifically recognizing PTI-1 protein underconditions permitting formation of complexes between the PTI-1 proteinand the antibody; and b) measuring the complex formed, thereby measuringthe level of PTI-1 protein expressed in the cancer cells.

[0025] This invention provides a method for detecting cancer cells in asample comprising steps of: a) isolating mRNA from the sample; b)contacting the isolated mRNA from step a with specific probe capable ofrecognizing a Prostate Tumor Inducing Gene under conditions permittingformation of a complex between the mRNA and the probe; and c) detectingof the complex formed wherein a positive detection of the complexindicates the presence of cancer cells in the sample.

[0026] This invention provides a method for determining whether asubject has metastatic or late stage prostate cancer comprising stepsof: a) obtaining an appropriate sample from the subject; and b)detecting the expression of a Prostate Tumor Inducing Gene in thesample, wherein a positive detection of the expression indicates thatthe subject has metastatic or late stage prostate cancer. Theappropriate sample includes but not limited to a blood, urine and semensample. The appropriate sample will contain prostate cancer cells suchthat the expression of Prostate Tumor Inducing gene may be detected.

[0027] In an embodiment, the Prostate Tumor Inducing Gene is PTI-1,PTI-2 or PTI-3.

[0028] In a specific embodiment of the above method, the expression isperformed by measuring the level of PTI-1 mRNA. In a further embodiment,the mRNA is measured by reverse transcription-polymerase chain reactionusing at least a pair of appropriate primers. In a still furtherembodiment, least one of the primers is selected from a group consistingof 5′GAGTCTGAATAGGGCGACTT3′, 5′AGTCAGTACAGCTAGATGCC3′,5′ACCCGAGAGGGGAGTGAAATA3′, 5′TGCCGCCATTCCACATTCAGT3′,5′ATGGGGGTAGAGCACTGAATG3′, 5′AACACCAGCAGCAACAATCAG3′ and5′AAATTAAGCTATGCAGTCGG 3′.

[0029] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0030] Experimental Details

[0031] Materials and Methods

[0032] Cell Lines. This study incorporated the following human celllines: prostate carcinoma (LNCaP, DU-145), breast carcinoma (T47D) andcolon carcinoma (SW480). Additional cell types studied include,CREF-Trans 6 cells and nude mouse tumor-derived CREF-Trans 6 cellstransfected with LNCaP DNA (CREF-Trans 6:4 NMT) (5). Cells were grown inDulbecco's modified Eagle's medium (DMEM) supplemented with 5% (rodentcells) or 10% (human cells) fetal bovine serum at 37° C. in a 95% air 5%CO₂— humidified incubator. All cell lines used in the present study weretested for mycoplasma contamination using the GenProbe mycoplasma testkit (Gaithersberg, Md.) and found to be mycoplasma free.

[0033] Genomic DNA Extraction and PCR. Human brain and kidneys werefrozen in liquid nitrogen, ground into powder, and digested with 100ug/ml proteinase K at 50° C. overnight, followed by phenol/chloroformextraction and ethanol precipitation (7,8). Oligonucleotides weresynthesized for PCR amplification corresponding to nt 147 to 167 UU(5′UTR Upper) and nt 550 to 570 UL (5′UTR Lower) of PTI-1 (GenBank,Accession no. L41490). Primer pair UU and UL will generate a 424 bpproduct. PCR was performed in a 50 ul volume, with 1 ug of brain orkidney genomic DNA, 0.5 uM of each primer (UU and UL), 400 uM dNTPs, 2mM Mg⁺⁺, and 1 unit of Taq DNA polymerase (GibcoBRL). Forty cycles ofamplification were performed with each cycle consisting of 1 min at 95°C., 1 min at 55° C. and 1 min at 72° C. on a programmable thermal cycler(MJ Research). “Hot start” PCR technique was applied. PCR products wereanalyzed on a 2% agarose gel by ethidium bromide staining.

[0034] RNA isolation from cultured cells and RT-PCR. Total cytoplasmicRNA was isolated from logarithmically growing cell cultures as describedpreviously (9,10). One ug of total RNA extracted from various tumor celllines was reverse transcribed into cDNA with 150 ng of random primersand 200 units of Superscript II RNase H—Reverse Transcriptase (GibcoBRL)in the presence of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMDTT and 500 mM dNTPs. The reaction mixture (20 ul) was incubated at 42°C. for 90 min, and terminated by heating at 70° C. for 15 min.Oligonucleotides were synthesized for RT-PCR amplification correspondingto nt 537 to 557 BU (Bridge Upper) and nt 768 to 788 BL (Bridge Lower)of the PTI-1 gene (GenBank, Accession no. L41490). Primer pair BU and BLwill generate a 252 bp product. PCR with 2 μl of the reversetranscription reaction mixture was similar to the genomic DNA protocolwith some modifications. Fifteen cycles of amplification were performedin the first round of PCR with primer BU (0.5 uM) alone, and then BL(0.5 uM) primer was added for another 40 cycles of amplification. PCRdirect sequencing (New England Biolabs) was performed with [γ-³²P]ATPlabeled primers following the manufacturer's recommendations.

[0035] Southern Blotting Analysis of RT-PCR Products of PTI-1 GeneTranscripts. Oligonucleotides were synthesized for southern blottinganalysis with the BSP (Bridge Specific Probe), 5′ AAATTAAGCTATGCAGTCGG3′. Ten pmol of the BSP oligonucleotide was incubated with 5 ul[γ-³²P]ATP (10 mCi/ml) and 20 units of T4 polynucleotide kinase(GibcoBRL) at 37° C. for 60 min in the presence of 70 mM Tris-HCl (pH7.6), 10 mM MgCl₂₁ 100 mM KCl and 1 mM β-mercaptoethanol. The reactionwas terminated by heating at 70° C. for 10 min, and labeledoligonucleotide probe was purified by ethanol precipitation. RT-PCRproducts amplified by the BU and BL primer pair were transferred ontonylon membrane (Hybond film, Amersham) according to standard capillaryblotting protocols. After fixation at 80° C. for 2 hr, the membrane wasincubated at 56° C. overnight in the presence of 1% SDS, 1 M NaCl and100 ug/ml sonicated salmon sperm DNA. Hybridization was performed thenext day by incubating with 1% SDS, 1M NaCl and labeled probe at 56° C.overnight. The membrane was washed twice with 100 ml 2×SSC at roomtemperature for 5 min each, once with 200 ml 2×SSC, 1% SDS at 56° C. for30 min, and once with 200 ml 0.1% SSC at room temperature. The blot wasexposed with Kodak X-OMAT film for 30 min at room temperature.

[0036] Determination of PCR Sensitivity. RNA was isolated from LNCaPcells and from mixtures of LNCaP and CREF-Trans 6 cells ranging from1:1000 to 1:100000000 as previously described (9,10). PCR was performedusing PTI-1 5′ UTR specific primers (5′-GAGTCTGAATAGGGCGACTT-3′ and5′-AGTCAGTACAGCTAGATGCC-3′) (7) and PSA specific primers(5′-TACCCACTGCATCAGGAACA-3′ and 5′-CCTTGAAGCACACCATTACA-3′) (14). TheCREF-Trans 6 cell line was chosen to dilute LNCaP cells because thiscell line does not express PTI-1 or PSA (7,8).

[0037] Patient Blood Samples, RNA Processing and PCR. Blood samples usedin this study were obtained from patients at Columbia-PresbyterianHospital and Mount Sinai Medical Center. Specimens were obtained withinformed consent of each patient using protocols approved by theInstitutional Review Boards of each respective hospital. Sample analysisincluded blood samples from 9 patients with stage D disease (3 with D2and 6 with D3), 12 patients with localized cancer of the prostate, 3healthy males and 3 healthy females. Venous blood (5 cc) was collectedin ethylenediaminetetracetic acid (EDTA) treated collection tubes,placed on ice and processed within 3 hr of phlebotomy (11). Samples werediluted in an equal volume of PBS and layered onto 8 cc ofFicoll-Plaque. The samples were centrifuged at 400×gravity for 30 minand the buffy coat cells were recovered. The cells were washed in PBSbefore RNA extraction. RNA was extracted as previously described (11,14) using a modified guanidinium thiocyanate/phenol/chloroformextraction technique (11) using the RNazole B reagent. Selected sampleswere analyzed for PSA and PSM expression by PCR using previouslydescribed techniques and primers (7,10,14). PTI-1 expression wasevaluated using the same 5′ UTR primer pair used for determining PCRsensitivity (7). Positive and negative PTI-1 expression was alsoconfirmed in a subset of samples using PCR with the UU and UL and the BUand BL primer pairs.

[0038] Experimental Results

[0039] The 5′ UTR Sequence of the PTI-1 Gene is Present in the HumanGenome. If PTI-1 is truly an etiologic agent in human prostate cancer,then this gene or related DNA sequences must be a component of humangenetic material. Demonstration that the 5′ UTR of PTI-1, which displaysa strikingly high degree of homology with prokaryotic ribosomal RNAsequences, is indeed present in the human genome was deemed a priorityfor future mechanistic studies of the PTI-1 gene. Moreover, thisinformation is required before and irrespective of the mechanism bywhich activation of this sequence occurs during cancer development.

[0040] As shown in FIG. 2, primer pair UU and UL (representing sequencesin the 5′ UTR of PTI-1) generated a specific PCR product with theexpected size (424 bp) from human brain genomic DNA. The size of the PCRproduct from genomic DNA (FIG. 2, Lanes 1 and 2) is the same size asthat of the PTI-1 cDNA suggesting that it is an intron free region. Itis well documented that both prokaryotic and eukaryotic ribosomal RNAgenes are without introns and the findings reported here are consistentwith this conclusion (12). RNase digestion of the template did notaffect the detection of this product. The amplification of this productalso required the simultaneous presence of all the following componentsin the PCR reaction mixture: Taq polymerase, both primers and thetemplate DNAs (FIG. 2). Similar experimental results occur when humankidney genomic DNA is substituted for brain genomic DNA (data notshown).

[0041] Although the present studies cannot rule out definitively thepossibility of a minute quantity of bacterial DNA in the genomic DNApreparations, thereby generating false positive results, it isrecognized that the probability of Mycoplasma contamination is lesslikely in tissue samples than in cell cultures (13). The possibilitythat mycoplasma contamination is present in the experimental reagents isnot likely at the level of sensitivity currently used to detect PTI-1 inexperimental DNA samples. These results support the conclusion that the5′ UTR sequence of the PTI-1 gene is actually a normal component of thehuman genome.

[0042] Detection of Junction Sequences Located Between the 5′ UTR andthe EF-1α Coding Region of the PTI-1 Gene in Total RNA from Tumor CellLines. It is hypothesized that even if there is Mycoplasma or relatedbacterial contamination in the cell cultures studied, this contaminationalone could not explain the presence of both prokaryotic ribosomal RNAsequences and human EF-1α sequences contiguous on the same RNA molecule.Thus, if such a junction point can be demonstrated to exist in totalRNA, it is unlikely that the identification of PTI-1 gene was due to anexperimental artifact.

[0043] As shown in FIG. 3 (panel A), primer pair BU (consisting ofsequences within the 5′ UTR of PTI-1) and BL (consisting of sequenceswithin the EF-1α region of PTI-1) generates an RT-PCR product with theexpected size (252 bp) in total RNAs from CREF-Trans 6:4 NMT (nude mousetumor-derived CREF-Trans 6 clone transfected with LNCaP HMW DNA), T47D(human breast carcinoma), SW480 (human colon carcinoma) and LNCaP andDU-145 (human prostate carcinoma) cells. This PCR product was notdetected in RNAs extracted from CREF-Trans 6 cells. This expressionpattern is identical to that previously reported using Northern blottinganalysis and probing with a PTI-1 5′ UTR-specific probe (7). As shown inpanel B, this PCR product also hybridizes with a PTI-1 cDNA specificoligonucleotide BSP. The BSP consists of 20 nucleotides, with 10nucleotides on either side of the junction point between the 5′ UTR andthe coding region of the PTI-1 cDNA (FIG. 1). The highly stringenthybridization and washing conditions (see Materials and Methods) used inSouthern blotting analysis demonstrates that this PCR product containsthe PTI-1 cDNA sequences, specifically the sequence surrounding thejunction point between the 5′ UTR and coding region of the PTI-1 gene.This conclusion was further supported by direct sequencing of one of thePCR products obtained from the tumor-derived CREF Trans 6:4 NMT cellline (data not shown), which documents that it consists of bothprokaryotic ribosomal-like RNA sequences and human EF-1α sequences.These sequences were the same as those published previously for thePTI-1 gene (7).

[0044] It was also important to rule out the possibility that the PTI-1plasmid might be a source of potential contamination generatingartifactual results. The PTI-1 gene was initially identified as a 1.8 Kbinsert from an LNCaP cDNA library. Subsequently, the remaining 215 bp atthe 5′ end was obtained by the RACE procedure (7). Since the missingregion of PTI-1 was located in the 5′ UTR, a full-length PTI-1 cDNA wasnever generated. Therefore, any PCR or RT-PCR products produced withprimers constructed from the first 215 bp and remaining 415 bp regionsof the 5′ UTR of PTI-1 could not be derived from a plasmid template, butcould only result from RNA or genomic DNA. As shown in FIG. 3 (panel C),primer pair uu (located inside the first 215 bp region that is missingin the PTI-1 cDNA clone) and UL (designed within the 5′ UTR of PTI-1that is present in the PTI-1 cDNA clone) permitted the amplification ofthe 5′ UTR sequences of PTI-1 from the same total RNAs of tumor celllines that were positive for the bridge region. Moreover, the pattern ofexpression of this sequence is identical to that of the junctionsequences of the PTI-1 gene (FIGS. 3A and B).

[0045] These results document that the PTI-1 gene is an authenticputative human oncogene that is expressed in specific human tumor celllines derived from appropriately transfected CREF-Trans 6 (CREF-Trans6:4 NMT) and prostate and additional human carcinomas.

[0046] Expression of the PTI-1 Gene in Prostate Carcinoma Patient BloodSamples. To determine the sensitivity of PTI-1 as a gene-based markerfor detecting prostate carcinoma cells, PTI-1 expressing LNCaP cellswere serially diluted with non-PTI-1 expressing CREF-Trans 6 cells,total RNA was isolated and samples were compared by RT-PCR for PTI-1expression (FIG. 4A). Using primers designed in the unique 5′ UTR regionof PTI-1, a positive PTI-1 specific amplified fragment (280 bp) wasdetected when 1 LNCaP cell was diluted in 108 CREF-Trans 6 cells. Incontrast, when primer sequences corresponding to PSA were employed inthe amplification, a weaker signal (corresponding to a 486 bp fragment)was obtained that represented 1 LNCaP cell diluted in 10⁶ CREF-Trans 6cells. The efficiency of detection of PSM (14) in serially diluted cellsusing a single pair of PSM-specific primers (generating a 647 bpfragment) was even less sensitive than PSA detecting 1 prostatecarcinoma cell diluted in 10⁵ CREF-Trans 6 cells (data not shown). Theseresults demonstrate that RT-PCR of PTI-1 is currently the most sensitivedetector of human prostate carcinoma cells available, significantlyexceeding the sensitivity of PSA and PSM.

[0047] The exquisite sensitivity of PTI-1 in detecting prostate cancercells in diluted samples (FIG. 4A) suggested that monitoring PTI-1transcripts might also prove useful as a direct screening test for thedetection of prostate carcinoma cells in the circulatory system ofprostate cancer patients. To determine if this assumption was correct,RT-PCR was performed using primers specific for the 5′ UTR of PTI-1 withRNAs isolated from blood samples confirmed as positive or negative forPSA and/or PSM expression (FIG. 4B). PTI-1 was able to detect carcinomacells in samples found to be positive for both PSA and PSM as well assamples found to be positive for only one of these two markers. Incontrast, 6 confirmed negative samples from volunteer females and malesand 4 patients with prostate cancer that had not spread past the marginof the prostate gland (also found negative for PSA and/or PSM) werenegative for PTI-1 expression (total of 18 samples: 8 of 8 confirmedpositives, 10 of 10 confirmed negatives) (FIG. 4B and unpublished data).

[0048] A second test of PTI-1 involved a double-blind study using 9random RNAs isolated from blood specimens. These samples were analyzedfor PSA and PTI-1 expression by RT-PCR using PSA-specific primers (11)and the primer pair UU and UL (within the 5′ UTR of PTI-1),respectively. Of these 9 samples, two were found to be positive forPTI-1 transcripts. These two samples were also positive for PTI-1 whenusing the BU and BL primer pair. One patient determined to havemetastatic prostate cancer, was positive for PTI-1 expression, butnegative for PSA expression. However, the other PTI-1 positive patientwas assumed by pathology to have localized cancer in the prostate glandand this patient's blood sample was also negative for RT-PCR of PSA. Inthe remaining 7 patients, 7 were confirmed as having non-metastaticprostate cancer and they were all negative for PTI-1 expression, whereas5 of 7 were negative and 2 were positive for PSA expression. The onlypatient who was suspicious for metastatic prostate cancer based on veryhigh serum PSA protein levels, was negative by RT-PCR for expression ofboth PTI-1 and PSA.

[0049] Taken together the studies described above indicate that 9 of 10(90% sensitivity) prostate cancer patients with confirmed metastaticdisease were positive for PTI-1 expression by RT-PCR, whereas only 4 of9 (44% sensitivity) blood samples from the same patients with metastaticdisease expressed PSA. Moreover, only 1 of 13 (˜8% potential falsepositive) patients without detectable signs of prostate carcinomaspreading from the prostate gland were positive for PTI-1, whereas 2 of10 (20% potential false positive) patients in the same group werepositive for PSA. Although further studies are needed with a largernumber of patient samples, including patients with and without confirmedmetastatic prostate cancer, the present studies provide compellingevidence suggesting that RT-PCR of PTI-1 should prove useful as asensitive methodology for monitoring extraprostatic disease in patientsprior to surgery, staging prostate cancer and evaluating patients'response to chemotherapy and radiation therapy.

[0050] Experimental Discussion

[0051] The ability to accurately monitor the aggressiveness of humanprostate cancer is a priority for defining the appropriate means oftherapeutically intervening in the progression of this disease. Recentstudies document that the identification of blood-borne PSA-expressingcells by RT-PCR can be achieved in patients with localized as well asmetastatic prostate cancer and that this detection provides a dependablemarker for predicting local invasion of a prostate tumor precedingsurgical procedures (11,14,15). PSA, a 34 kDa glycoprotein, is aprostate-associated serine protease with predominant expression byprostate epithelial cells, the cells most often associated withprostatic oncogenesis (16,17). Recently, assays used to monitor thisprotein in the blood have changed the management of prostate cancerpatients by permitting the early detection of prostate tumors as well asby providing a more efficient means to follow the progression of thisdisease. The specificity of PSA for prostate cells has permitted thedevelopment of an RT-PCR based assay that can detect as few as 1PSA-synthesizing cell in ₁₀′ non-PSA expressing blood cells (11,14,15).A further enhancement of the PSA assay involves the addition ofdigoxigenin-modified nucleotide to the PCR reaction (9,13). A Southernblot made from such electrophoreses reaction products can then beanalyzed by sensitive immunostaining techniques that greatly enhance thedetection of the specific PSA-derived cDNA product. When this enhancedassay was previously applied to peripheral blood specimens taken fromprostate cancer patients with confirmed metastases, it was positive forthe majority (77.7%) of patients in this category that were studied(9,13). PTI-1 is not expressed in normal prostate or BPH, but PTI-1expression is apparent in both PSA-positive hormone-sensitive, LNCaP,and PSA-negative hormone-refractive, DU-145, human prostate carcinomacells (7). The current study demonstrates a ≧100-fold increase in thesensitivity of RT-PCR of PTI-1 versus PSA and PSM in detecting prostatecancer cells. Moreover, PTI-1 RT-PCR resulted in an ˜90% sensitivity indetecting patients with metastatic prostate cancer versus an ˜44%sensitivity with the same patient samples using RT-PCR with PSA. Inthese contexts, an RT-PCR-based assay using PTI-1 may permit thedetection of prostate cancer cells in the circulation that would not bedetected using RT-PCR with PSA or PSM. In addition, the specificity ofPCTA-1 for prostate carcinoma versus normal prostate or BPH eliminatesthe possibility of generating false positives that could occur usingRT-PCR of PSA or PSM.

[0052] Important questions that were experimentally addressed includethe nature of the 5′ UTR region of PTI-1 that displays homology tobacterial ribosomal 23S RNA and the genuineness of the PTI-1 gene (7).The PTI-1 gene was cloned from an LNCaP human prostate cancer cDNAlibrary using a 214 bp fragment detected by differential RNA display inLNCaP DNA transfected tumor-derived CREF-Trans 6 cells (7). Sequenceanalyses confirm that the 214 bp sequence with homology to prokaryoticribosomal RNA is located within the 5′ UTR of the PTI-1 gene (7).Although all of the cell lines used in the original study were testedusing the GenProbe mycoplasma test kit and found to be free ofmycoplasma contamination, it is still possible that the PTI-1 gene arosefrom a cloning artifact generated by a low level of undetected bacterialcontamination present in the CREF-Trans 6:4 NMT or LNCaP cell lines.Several lines of evidence are presently provided that argue against thispossibility and validate the authenticity of the PTI-1 gene. UsingPCR-based approaches with genomic DNAs isolated from human brain andkidney, the presence of the prokaryotic ribosomal RNA homologoussequences in the human genome is doucmented by this study. Using anRT-PCR-based strategy with primers corresponding to the 5′ UTR and EF-1αcoding region of PTI-1, an appropriate junction sequence is amplifiedfrom total RNA from CREF Trans 6:4 NMT cells, various human tumor celllines and blood samples from patients with confirmed metastatic prostatecancer. Even if the tissue samples and cell lines contained mycoplasmaor related bacterial contamination, these adventitious organisms alonewould not be capable of generating the unique junction sequence in apopulation of total cytoplasmic RNAs. The ability to detect such ajunction sequence in total RNAs from specific cancer cells providescompelling evidence for the authenticity of the PTI-1 gene and its'encoded message.

[0053] The present study raises a number of important issues thatrequire resolution in order to define the role of PTI-1 in human canceretiology and evolution. Documentation of the presence of the 5′ UTRmycoplasma homology region in the human genome forces one to examine thepotential origin of such prokaryotic gene sequences in the eukaryoticgenome. Although no mechanism is provided, a recent study suggests apotential relationship between persistent chronic infection withmycoplasmas and malignant transformation (18). Unlike retroviruses andDNA tumor viruses which can incorporate their genetic material into thehost genome, no evidence is currently available indicating thatmycoplasma gene sequences can integrate into the human genome as part oftheir infectious cycle. However, it is possible that Mycoplasma, or morelikely one of its ancestors, may randomly insert its genetic materialinto human or one of its ancestor's genome. This integration may occurby a mechanism that is similar to that by which foreign gene sequencesinsert into the genome of transgenic animals. The presence of sequencesthat are highly homologous to prokaryotic genes in the human genome hasbeen previously reported, although the functions of these sequences arenot known (19). It is very tempting to speculate that, based on the highdegree of homology between the 5′UTR of PTI-1 and mycoplasma genesequences, such an event may have occurred recently in evolution,thereby generating the PTI-1 gene.

[0054] Additional important issues are the mechanism by which PTI-1expression is activated in human tumor cells and the role of PTI-1 inmediating the cancer phenotype. Differential expression of PTI-1 incancer cells may occur by activation of transcription from an upstreampromoter from the EF-1α or another target gene, resulting intranscription of the 5′UTR and EF-1α region of PTI-1. Alternatively,gene activation could result from genome rearrangement, including genedeletion, inversion and translocation, which are common occurrences inmany cancers (20,21). Elucidation of the genomic structure, includingthe promoter region, of PTI-1 is necessary to shed light on thisquestion and to define the molecular basis for the differentialexpression of PTI-1 in human prostate, and additional, cancers versusnormal cells. Further studies are also necessary to determine thefunctional relevance of PTI-1 expression in determining the cancerphenotype. If PTI-1 expression is shown to be causally related to cancerdevelopment or progression, then this gene could serve as a potentialtarget for inhibiting the neoplastic process.

[0055] Second Series of Experiments

[0056] Bloods were screened for the presence of PTI-1 using a previouslydescribed protocol (Sun, et al. Cancer Research 57:18-23, 1997).Briefly, total RNA was reverse transcribed into cDNA with 150 ng ofrandom primers and 200 U of Superscript II RNAase H′ (Life Technologies)in the presence of 50 mM Tris-HCL pH8.3, 75 mM KCI, 3 mM MgCI2, 10 mMDTT, and 500 uM deoxynucleotide triphosphates. The reaction mixture (20ul) was incubated at 42 C for 90 minutes and terminated by heating at 70C for 15 minutes. Oligonucleotides were synthesized for RT-PCRamplification according to sequence of the PTI-1 gene (GenBank accessionno. L41490). The following primer pairs were used: Primer UU  (5′ UTR5′ ACCCGAGAGGGGAGTGAAATA 3′ Upper) Primer UL  (5′ UTR5′ TGCCGCCATTCCACATTCAGT 3′ Lower) Primer BU  (Bridge5′ ATGGGGGTAGAGCACTGAATG 3′ Upper) Primer BL  (Bridge5′ AACACCAGCAGCAACAATCAG 3′ Lower) Oligonucleotide5′ AAATTAAGCTATGCAGTCGG 3′ BSP  (Bridge Specific Probe)

[0057] Experimental Results:

[0058] All mRNAs were screened for degradation by electrophoresis (notshown) and for the presence of the housekeeping gene, GAPDH, by RT-PCR(FIG. 1). Amplification with the BU and BL primers is shown in FIG. 2.As can be seen, a large number of blood RNAs were positive for thepresence of the bridge region. A sample from an individual with noevidence of cancer had a very light band. This gel was blotted andsubsequently probed with a 20 mer bridge specific primer (BSP). As canbe seen in Table 1, 14 of 33 samples were positive. TABLE 1 Bloodsamples positive for the bridge (BU and BL) and the bridge specific(BSP) primers SAMPLE BSP BU/BL  1 Neg Neg  2 + Pos  3 Neg Pos  4 Neg Neg 5 Neg Neg  6 +++ Pos  7 Neg +/−  8 +++ Pos  9 Neg Neg 10 Neg Pos 11 +Pos 12 Neg Pos 13 +++ Pos 14 Neg Pos 15 +++ Pos 16 Neg Pos 17 Neg Neg 18Neg Neg 19 +++ Pos 20 Neg Pos 21 Neg Pos 22 +++ Pos 23 Neg Neg 24 NegPos 25 Neg Pos 26 ++ Pos 27 Neg Pos 28 Neg Neg 29 ++ Pos 30 +++ Pos 31+++ Pos 32 + +/− 33 Normal Sample Neg +/−

[0059] The normal blood sample (#33) was negative for the BSP.Additional screening of the blood samples was done using the 5′ UTRprimers UU and UL. It required two rounds of amplification to visualizethe bands. As can be seen, 4 of 33 samples had a band in the appropriatebp region (FIG. 3). Two additional samples had a band slightly higherand slightly lower than the expected molecular weight. The normal bloodsample was negative for the 51′ UTR.

[0060] Experimental Discussion

[0061] The results of these experiments confirm the earlier utility ofPTI-1 as a diagnostic for late stage prostate cancer. All of thepositive samples (using the BSP and the 5′ UTR) had previously beendiagnosed as having late stage or metastatic disease. Interestingly, twobands were evident using the 5′ UTR primers that differed from theexpected molecular weight for PTI-1. Since this gene is a member of alarge family of related sequences, it is possible that other PTIs mayhave diagnostic utility for cancer detection.

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1 11 1 21 DNA Artificial Sequence Synthetic oligonucleotide 1 acccgagaggggagtgaaat a 21 2 21 DNA Artificial Sequence Synthetic oligonucleotide 2tgccgccatt ccacattcag t 21 3 21 DNA Artificial Sequence Syntheticoligonucleotide 3 atgggggtag agcactgaat g 21 4 21 DNA ArtificialSequence Synthetic oligonucleotide 4 aacaccagca gcaacaatca g 21 5 20 DNAArtificial Sequence Synthetic oligonucleotide 5 aaattaagct atgcagtcgg 206 20 DNA Artificial Sequence Synthetic oligonucleotide 6 gagtctgaatagggcgactt 20 7 20 DNA Artificial Sequence Synthetic oligonucleotide 7agtcagtaca gctagatgcc 20 8 20 DNA Artificial Sequence Syntheticoligonucleotide 8 tacccactgc atcaggaaca 20 9 20 DNA Artificial SequenceSynthetic oligonucleotide 9 ccttgaagca caccattaca 20 10 1819 DNA HomoSapiens 10 cggaccgagc tccgttgcat tttgatgaat ccatagtcaa attagcgagacacgttgcga 60 attgaaacat cttagtagca acaggaaaag aaaataaata atgatttcgtcagtagtggc 120 gagcgaaagc gaaagagccc aaacctgtaa aggggggttg gtaggacatcttacattgag 180 ttacaaaatt ttatgatagt agaagaagtt gggaaagctt caacatagaaggtgatattc 240 ctgtatacga aatcataaaa tctcatagat gtatcctgag tagggcggggtacgtgaaac 300 cctgtctgaa tctgcccggg accacccgta aggctaaata ctaatcagacaccgatagtg 360 aactagtacc gtgagggaaa ggtgaaaaga acccgagagg ggagtgaaatagattctgaa 420 accatttact tacaagtagt cagagcacgt taaagtgtga tggcgtacatcttgcagtat 480 gggccggcga gttatgttaa tatgcaaggt taagcacgaa aaaagcggagccgtagggaa 540 accgagtctg aatagggcga ctttagtata ttggcatata cccgaaaccaggtgatcatc 600 catgagcagg ttgaagctta ggtaaaacta agtggaggac cgaaccgtagtacgctaaaa 660 agtgcccgga tgacttgtgg atagtggtga aattccaatc gaacctggagatagctggtt 720 ctcttcgaaa tagctttagg gctagcgtat agtactgttt aatgggggtagagcactgaa 780 tgtggaatgg cggcatctag ctgtactgac tataatcaaa ctccgaataccattaaaatt 840 aagctatgca gtcggaacgt gggtgataac gtccacgctc gcgagggaaacaacccagat 900 ccgtcagcta aggtcccaaa aatgtgttaa gtgagaaagg ttgtggagatttcataaaca 960 actaggaagt tggtttagaa gcagccacct tttaaagagt gcgtaattgctcactagtca 1020 agagatcttg cgccaataat gtaacgggac tcaaacacaa tacccaagctacgggcacat 1080 tatgtgcgtt aggagagcgt tttaatttcg ttgaagtcag accgtgagactggtggagag 1140 attaaaagtt cgagaatgcc ggcatgagta acgattcgaa gtgagaatcttcgacgccta 1200 ttgggaaagg tttcctgggc aaggttctcc acccagggtt agtcagggcctaagatgagg 1260 cagaaatgca tagtcgatgg acaacaggtt aatattcctg tacttggtaaaagaatgatg 1320 gagtgacgaa aaaggatagt tctaccactt ccactatgtc ctatcaataggagctgtatt 1380 tggcatcata ggaggcttca ttcactgatt tcccctattc tcaggctacaccctagacca 1440 aacctacgcc aaaatccatt tcactatcat attcatcggc gtaaatctaactttcttccc 1500 acaacacttt ctcggcctat ccggaatgac ccgacccgac gttactcggactaccccgat 1560 gcatacacca catgaaacat cctatcatct gtaggctcat tcatttctctaacagcagta 1620 atattaataa ttttcatgat ttgagaagcc ttcgccttcg aagcgaaaagtcctaatagt 1680 agaagaaccc tccataaacc tggagtgact atatggatgc ccccaccctacctcacattc 1740 gaagaacccg tatacataaa atctagacaa aaaaggaagg aagtgaacgccccacaaaaa 1800 aaaaaaaaaa aaaaaaaaa 1819 11 1869 DNA Homo Sapiens 11aactaagtgg aggaccgaac cgtagtacgc taaaaagtgc ccggatgact tgtggatagt 60ggtgaaattc caatcgaacc tggagatagc tggttctctt cgaaatagct ttagggctag 120cgtatagtat tgtttaatgg gggtagagca ctgaatgtgg aatcggcggc atctagctgt 180actgactata atcaaactcc gaataccatt aaaattaagc tatgcagtcg gaacgtgggt 240gataacctcc actctcgcga gggaaacaac ccagatcgtc agctaaggtc ccaaaattgt 300gttaagtgag aaaggttgtg agatttcata aacaactagg aagttggctt agaagcagcc 360accttttaaa gagtgcgtaa ttgctcacta gtcaagagat cttgcgccaa taatgtaacg 420ggactcaaac acaataccga agctacgggc acattatgtc ggttaggaga gcgttttaat 480ttcgttgaag tcagaccgtg agactggtgg agagattaaa agttcgagaa tgcccggcat 540gagtaacgat tcgaagtgag aatcttcgac gcctattggg aaaggtttcc tgggcaaggt 600tcgtccaccc agggttagtc agggcctaag atgaggcaga aatgcatagt cgatggacaa 660caggttaata ttcctgtact tggtaaaaga atgatggagt gacgaaaaag gatagttcta 720ccacttactg gattgtgggg taagcaacaa gagagttata taggcaaatc cgtatagcat 780aatcttgagt tgtgatgcat agtgaagact tcggtcgagt aacgaattga atcgatttca 840tgtttccaag aaaagcttct agtgttaatt ttttatcaac ctgtaccgag aacgaacaca 900cgttcccaag atgagtattc taaggcgagc gagaaaacca atgttaagga actctgcaaa 960ataaccccgt aagttcgcga gaaggggcgc ctatttttaa taggccacag aaaatagggg 1020ggcaactgtt tatcaaaaac acagctctct gctaagttgt aaaacgacgt atagagggtg 1080aagcctgccc agtcccgaag ttaaacggag atgttagctt acgcaaagca ttaaagtgaa 1140gcccgggtga acggcggccg taactataac ggtcctaagg tagcgaaatt ccttgtcaac 1200taattattga cctgcacgaa aggcgcaatg atctccctac tgtctcaaca ttggactcgg 1260tgaaattatg gtaccagtga aaacgcaggt tacccgcatc aagacgaaaa gaccccgtgg 1320agctttacta taacttcgta ttgaaaattg gtttagcatg tgtaggatag gcgggagact 1380ttgaagctgg gacgctagtt ctagtggagt caaccttgaa ataccaccct tgctaaattg 1440attttctaac ccgttcccct tatctggaag gagacagtgc gtggtgggta gtttgactgg 1500gcggtcgcct cctaaagtgt aacggaggcg ttcaaagcta cactcaatat ggtcagaaac 1560catatgcaga gcacaaaggt aaaagtgtgg ttgactgcaa gacttacaag tcgagcaggt 1620gcgaaagcag gacttagtga tccggcggta cattgtggaa tggccgtcgc tcaacggata 1680aaagtcaccc cggggataac aggctaatct tccccaagag atcacatcga cgggaaggtt 1740tggcacctcg atgtcggctc atcgcatcct ggagctggag tcggttccaa gggtttgctg 1800ttcgccaatt aaagcggtac gtgagctggg ttcagaacgt cgtgagacag ttcggtcctc 1860cacttagtt 1869

What is claimed:
 1. An isolated mammalian nucleic acid comprising SEQ IDNO:10.
 2. An isolated nucleic acid comprising at least 15 nucleotidesthat hybridizes to SEQ ID NO:10 or a nucleic acid complementary theretounder stringent hybridization conditions.
 3. The nucleic acid of claim 2operatively linked to a promoter of RNA transcription.
 4. A vectorcomprising the nucleic acid of claim
 3. 5. The plasmid of claim
 4. 6.The plasmid of claim 5 having ATCC Accession No.
 69742. 7. A method fordetecting cancer cells in a sample comprising detection of theexpression of Prostate Tumor Inducing Gene-2 in the sample, comprisingthe steps of: (a) preparing RNA from the sample; (b) performing reversetranscription-polymerase chain reaction under conditions that permitcopies of Prostate Tumor Inducing Gene-2 nucleic acid to be produced,using primers specific for said Prostate Tumor Inducing Gene-2; and (c)determining whether a detectable amount of product is produced by thereverse transcription-polymerase chain reaction; wherein a detectableamount of product constitutes a positive detection of expression, apositive detection of expression indicates the presence of cancer cellsin the sample, and said Prostate Tumor Inducing Gene-2 comprises SEQ IDNO:10.
 8. A method for determining whether a subject has metastatic orlate stage prostate cancer comprising the steps of: (a) obtaining anappropriate sample from the subject; (b) preparing RNA from the sample;(c) performing reverse transcription-polymerase chain reaction underconditions that permit copies of Prostate Tumor Inducing Gene-2 nucleicacid to be produced, using primers specific for the Prostate TumorInducing Gene-2; and (d) determining whether a detectable amount ofProstate Tumor Inducing Gene-2 product is produced by the reversetranscription-polymerase chain reaction; wherein a detectable amount ofproduct constitutes a positive detection of expression, a positivedetection of expression indicates that the subject has metastatic orlate stage prostate cancer, and said Prostate Tumor Inducing Gene-2comprises SEQ ID NO:10.
 9. An isolated mammalian nucleic acid comprisingSEQ ID NO:11.
 10. An isolated nucleic acid comprising at least 15nucleotides that hybridizes to SEQ ID NO:11 or a nucleic acidcomplementary thereto under stringent hybridization conditions.
 11. Thenucleic acid of claim 10 operatively linked to a promoter of RNAtranscription.
 12. A vector comprising the nucleic acid of claim
 11. 13.The plasmid of claim
 12. 14. The plasmid of claim 12 having ATCCAccession No.
 69742. 15. A method for detecting cancer cells in a samplecomprising detection of the expression of Prostate Tumor Inducing Gene-3in the sample, comprising the steps of: (a) preparing RNA from thesample; (b) performing reverse transcription-polymerase chain reactionunder conditions that permit copies of Prostate Tumor Inducing Gene-3nucleic acid to be produced, using primers specific for said ProstateTumor Inducing Gene-3; and (c) determining whether a detectable amountof product is produced by the reverse transcription-polymerase chainreaction; wherein a detectable amount of product constitutes a positivedetection of expression, a positive detection of expression indicatesthe presence of cancer cells in the sample, and said Prostate TumorInducing Gene-3 comprises SEQ ID NO:11.
 16. A method for determiningwhether a subject has metastatic or late stage prostate cancercomprising the steps of: (a) obtaining an appropriate sample from thesubject; (b) preparing RNA from the sample; (c) performing reversetranscription-polymerase chain reaction under conditions that permitcopies of Prostate Tumor Inducing Gene-3 nucleic acid to be produced,using primers specific for the Prostate Tumor Inducing Gene-3; and (d)determining whether a detectable amount of Prostate Tumor InducingGene-3 product is produced by the reverse transcription-polymerase chainreaction; wherein a detectable amount of product constitutes a positivedetection of expression, a positive detection of expression indicatesthat the subject has metastatic or late stage prostate cancer, and saidProstate Tumor Inducing Gene-3 comprises SEQ ID NO:11.
 17. A method fordetecting cancer cells in a sample comprising detection of theexpression of Prostate Tumor Inducing Gene-i in the sample, comprisingthe steps of: a) contacting the sample with an antibody capable ofspecifically recognizing PTI-1 protein under conditions permitting theformation of a complex between the PTI-1 protein and the antibody; andb) measuring the complex formed, thereby detecting cancer cells in thesample.
 18. The method of claim 17, wherein the antibody is a monoclonalantibody.